Objectives This study aimed to investigate diagnostic value of monocyte-to-lymphocyte ratio as a novel biomarker in children with appendicitis and to compare with other biomarkers. Methods A total of 683 children were included in this retrospective study. Patients were categorized as acute appendicitis (AA, n = 254), perforated appendicitis (PA, n = 82), nonspecific abdominal pain (NAP, n = 197), and control (n = 150). The groups were compared for demographics, duration of symptoms, monocyte-to-lymphocyte ratio (MLR), platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), white blood cell count, neutrophil percentage (NP), C-reactive protein (CRP), mean platelet volume, and sodium level. Results There was a significant difference between all groups for MLR, PLR, and CRP. White blood cell count, NP, and NLR were significantly increased in patients with appendicitis, but there was no significant difference in the differential diagnosis between PA and AA. Sodium levels significantly decreased in patients with appendicitis. No statistically significance was determined between all groups in term of mean platelet volume. Among the biomarkers, MLR showed the highest diagnostic value (area under the receiver operator characteristic curve = 0.798) for AA, while CRP showed excellent diagnostic value (area under the receiver operator characteristic curve = 0.897) for PA. Conclusions To our knowledge, this is the first study showing that MLR is a valuable parameter to differentiate patients with and without appendicitis. White blood cell, NP, and NLR are also useful parameters. On the other hand, an increased CRP level and hyponatremia are indicators of PA.